CN109839508B

CN109839508B - Application of Rbm24-S181 site phosphorylation as marker of drug for mental stress diseases and related heart diseases

Info

Publication number: CN109839508B
Application number: CN201910089104.7A
Authority: CN
Inventors: 张敏; 杨鹤; 孙雷
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2019-01-30
Filing date: 2019-01-30
Publication date: 2021-06-29
Anticipated expiration: 2039-01-30
Also published as: CN109839508A

Abstract

The invention relates to the field of biomedicine, and provides application of Rbm24-S181 site phosphorylation as a marker for mental stress diseases and related cardiac disease medication in order to solve the problem that few and few diagnostic markers for cardiac diseases caused by mental stress.

Description

Application of Rbm24-S181 site phosphorylation as marker of drug for mental stress diseases and related heart diseases

Technical Field

The invention relates to the field of biomedicine, in particular to application of Rbm24-S181 site phosphorylation as a marker for mental stress diseases and related heart diseases.

Background

With the acceleration of life rhythm, social competition is increasingly violent, people have greater and greater life pressure, most people are running under overload, and mental stress diseases with different degrees, such as depression, anxiety, insomnia, mania and the like, are easy to generate. The diagnosis of mental diseases is very complicated, and the specific diagnosis of mental diseases at present fails to obtain ideal markers. Clinically, the diagnosis of mental disorders depends mainly on the clinical symptoms of the patient. However, with the development of medical level, in clinical diagnosis, the mental disease can be accurately diagnosed according to the mental symptoms and the results of biochemical examination, and the reduction of the damage of the drug to the organs of the patient by taking the drug according to the results of biochemical examination is also of positive significance. At present, biochemical detection indexes such as neurotransmitters, enzymes and cyclic nucleotides are mainly used clinically, and can be applied to auxiliary diagnosis of mental diseases, but the biochemical detection still has some limitations.

According to the literature, mental disorder diseases such as depression or anxiety are closely related to the occurrence of subsequent heart diseases. In patients with depression, the incidence of heart disease is higher than that in normal humans. But few and few diagnostic markers for the development of heart disease caused by mental stress.

Therefore, it is necessary to find a marker for diagnosing whether the risk of heart diseases of sub-healthy people suffering from mental stress is increased, so that mental disease patients can find whether heart diseases exist in early stage and can be treated in time.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides the application of Rbm24-S181 site phosphorylation as a biomarker in heart diseases caused by mental stress and mental stress, and provides a new target and an intervention strategy for molecular diagnosis for evaluating mental stress and heart diseases by Rbm24-S181 site phosphorylation (the amino acid sequence of Rbm24-S181 is shown as SEQ ID No.2, namely, Rbm24 protein 181 th serine).

The purpose of the invention is realized by the following technical scheme:

the Rbm24-S181 site phosphorylation is used as a biomarker in mental stress diseases.

The Rbm24-S181 site phosphorylation is used as a molecular marker in the diagnosis of heart diseases caused by mental stress.

Application of Rbm24-S181 site phosphorylation as drug target in prevention and treatment of heart diseases caused by related mental stress

The substance for detecting the phosphorylation level of the Rbm24-S181 locus is applied to the preparation of products for preventing, diagnosing, treating and diagnosing mental stress diseases and heart diseases caused by mental stress.

Preferably, the substance is a polyclonal antibody or a monoclonal antibody which recognizes phosphorylation of Rbm24-S181, and the amino acid sequence of the monoclonal antibody is YAApSPAAAGYVTA.

The principle of the invention is as follows:

rbm24 belongs to the RBM family of RNA binding proteins (amino acid sequence shown in the specification) and is specifically and highly expressed in heart. Rbm24 plays an important role in regulating cardiac development, and due to the high sequence similarity of Rbm24 among different species, the Rbm represents strong function conservation. In mice, Rbm24 is involved in early development of the heart, and Rbm24 gene deletion causes damage to the endocardial pad in mice, thereby causing embryonic death. In a molecular epidemiological study of human coronary heart disease, Rbm24 was identified as one of the potential biomarkers and major risk factors for coronary heart disease development, with transcriptional levels that were significantly up-regulated in coronary heart disease patients. Therefore, Rbm24 plays an important role in the development of heart disease.

Protein phosphorylation is one of the important mechanisms for post-translational modification of proteins, and is at the primary position in the cellular response to external signals. Previous work shows that phosphorylation at the Rbm24-S181 site of the heart responds to mental stress signals, and the regulation relation between mental stress and phosphorylation at the Rbm24-S181 site is explored to be used as a molecular marker for heart diseases caused by mental stress.

The invention has the beneficial effects that:

the invention utilizes Rbm24-S181 site phosphorylation, can realize the diagnosis of diseases through blood, has the advantages of convenience and rapidness, can relieve the pain of patients on one hand, and can also relieve the workload of medical care personnel on the other hand, thereby improving the working efficiency. The invention also belongs to a diagnosis method of a gene diagnosis type, has higher specificity, and enables patients to discover and treat early and control the state of illness in time.

Drawings

FIG. 1a is a diagram of an immunopeptide fragment prepared from the antibody used in the present invention;

FIG. 1b is a graph showing the western-blot assay for overexpression of 3Flag-Rbm24 in cardiomyocyte C2C 12.

FIG. 1c is a graph of the western-blot assay for knock-down of Rbm24 protein levels in colorectal cancer cells HCT 116.

FIG. 1d is a graph of the protein level of Rbm24 knocked out by the western-blot assay in colorectal cancer cells HCT116 using the criprpr-cas 9 technique

FIG. 2a is a graph showing the significant change in phosphorylation at Rbm24-S181 after epinephrine treatment in cardiomyocytes C2C12 in the western-blot assay, wherein lithium is an inhibitor of upstream kinase phosphorylation at the Rbm 24-S181.

FIG. 2b is a graph showing the significant change of phosphorylation at Rbm24-S181 site of mouse heart after the treatment of C57 mouse with intraperitoneal adrenaline injection, wherein lithium is an Rbm24-S181 site phosphorylation upstream kinase inhibitor.

FIG. 2C is a graph showing that the phosphorylation of Rbm24-S181 site of heart of mice is significantly changed after the mice are subjected to chronic stress treatment by using western-blot detection C57, wherein lithium is an Rbm24-S181 site phosphorylation upstream kinase inhibitor.

FIG. 3 is a significant change diagram of phosphorylation at Rbm24-S181 locus detected by serum obtained after blood purification after a western-blot C57 mouse is subjected to chronic pressure treatment at different times;

FIG. 4 shows the amino acid sequence of Rbm24 (the grayscale portion is the RNA binding region).

Detailed Description

The present invention is described in further detail below with reference to specific examples so as to be understood by those skilled in the art.

Example 1: preparing an Rbm24-S181 site phosphorylation antibody and verifying that the antibody can be used

FIG. 1 a-FIG. 1D identify Rbm24-S181 site phosphorylation, and then immunize rabbits according to the peptide fragment shown in FIG. 1a, a monoclonal antibody specifically recognizing Rbm24-S181 phosphorylation is prepared, the amino acid sequence of the monoclonal antibody is YAApSPAAAGYVTA, the peptide fragment is highly conserved in animals such as human, mouse (YAApSPAAAGYVTT) and zebra fish (YAApS PAAT GYVA A, shown in SEQ ID No. 1), and the monoclonal antibody can recognize Rbm24 and S181D mutants (aspartic acid D can simulate phosphorylation) which are overexpressed in cells, but does not recognize S181A (alanine A can simulate non-phosphorylation) mutants.

This result demonstrates that S181D mimics S181 phosphorylation and is specifically recognized by phosphorylated antibodies. Meanwhile, the Rbm24-S181 phosphorylation antibody can specifically recognize in vivo expression phosphorylation Rbm24, and by applying siRNA technology, the expression level of Rbm24 in cells is reduced, and the phosphorylation level of Rbm24-S181 is also reduced. The gene of Rbm24 in cells is knocked out by applying a criprpr-cas 9 technology, and Rbm24-S181 phosphorylation cannot occur.

Example 2: phosphorylation of Rbm24-S181 site in response to stress

The experimental conditions in FIG. 2a were such that a protein sample was collected after 30min of treatment with epinephrine at a final concentration of 40uM in mouse cardiomyocytes C2C 12. The results of the western-blot analysis are shown in FIG. 2a, where Rbm24-S181 was significantly activated for phosphorylation (lane 3). Lanes 2 and 4 are the Lithium group, where lane 2 shows a significant decrease in the background level of Rbm24-S181 phosphorylation when Lithium alone is added, and lane 4 shows a significant inability to activate the level of Rbm24-S181 phosphorylation after 30min of the addition of epinephrine.

FIG. 2b shows the experimental conditions that C57 male mice were intraperitoneally injected with 5mg/kg epinephrine for 1h and then sacrificed with carbon dioxide, and the cardiac samples were examined by western-blot, and the results are shown in FIG. 2b, in which Rbm24-S181 phosphorylation was significantly activated (lane 3). Lanes 2 and 4 are the intraperitoneal Lithium group, where lane 2 shows a significant decrease in the background level of Rbm24-S181 phosphorylation when only Lithium is injected, and lane 4 shows a significant inability to activate the level of Rbm24-S181 phosphorylation when both Lithium and epinephrine are injected.

The experimental conditions in FIG. 2C were such that C57 male mice were raised in isolation, i.e., one mouse was housed in a cage, for 30 days, and after 30 days, the mice were sacrificed by co2 and the hearts were examined by western blot, the results of which are shown in FIG. 2C, and Rbm24-S181 phosphorylation was significantly activated (lane 3). Lanes 2 and 4 are the i.p. Lithium group, where lane 2 is the r.p. Lithium injected alone per day and the background level of Rbm24-S181 phosphorylation was significantly reduced in the population, and lane 4 is the r.p. Lithium injected per day and isolated for 30 days per day and the r.p. m24-S181 phosphorylation was significantly not activated.

Example 3: detection of Rbm24-S181 phosphorylation by mouse serum

The experimental conditions of fig. 3 are that the male C57 mouse is isolated, the blood of the mouse is taken after 10 days, 20 days, 30 days and 40 days of isolation, the blood is stood for a period of time and then centrifuged to obtain serum, and the serum is subjected to western-blot detection. As shown in FIG. 3, the Western-blot results show that the phosphorylation levels of Rbm24-S181 in the serum of the mice subjected to chronic stress at different times are obviously increased in a gradient manner.

In combination with the above examples 1-3, the present invention can be used for diagnosing, preventing and treating mental stress such as depression, anxiety, insomnia, etc., and mental stress-induced heart diseases such as cardiac hyperplasia, cardiac hypertrophy, cardiac fibrosis, heart valve injury, etc., based on the phosphorylation level of Rbm24-S181 site, and the phosphorylation of Rbm24-S181 site is used as a molecular marker in diagnosing mental stress and mental stress-induced heart diseases.

Other parts not described in detail are prior art. Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and the embodiments are within the scope of the present invention.

Sequence listing

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Claims

1. An epitope peptide which recognizes a phosphorylated monoclonal antibody by Rbm24-S181, characterized in that: the amino acid sequence of the epitope peptide of the monoclonal antibody is YAApSPAAAGYVTA.